Presence of cholesteryl ester-laden macrophage foam cells is one of the most prominent characteristics of an atherosclerotic lesion. Extracellular acceptor-mediated free cholesterol (FC) efflux is the primary mechanism for the removal of cellular cholesterol and is critical in preventing foam cell formation and atherosclerotic lesion development. However, in order for efflux to occur, FC must first be released from stored cholesteryl esters (CE) by hydrolysis mediated by neutral cholesteryl ester hydrolase (CEH) and this step is increasingly being recognized as the rate-limiting step in FC efflux. The PI has cloned and characterized the human macrophage CEH and demonstrated increased cellular CE mobilization after CEH over-expression and establishing the role of CEH in atherogenesis by regulating cellular CE hydrolysis was the focus during the last funding cycle. Macrophage-specific transgenic expression of human macrophage CEH in athero-susceptible LDLR-/- mice not only led to a significant reduction in diet-induced atherosclerosis and cholesterol content of the aortic lesions but also reduced lesion necrosis. Our mechanistic studies showed that enhancing CEH-mediated hydrolysis of CE in macrophages enhanced the flux of cholesterol from macrophages to liver for the final elimination as bile acids into bile and feces providing direct evidence that enhanced CEH-mediated intracellular CE hydrolysis is anti-atherogenic. The overall objective of the current proposal is to expand on our work on the role of CEH in regulating macrophage lipid burden in three critical areas: to delineate the effect of enhancing macrophage CEH expression on progression of existing atherosclerotic lesions; to determine the systemic effects of CEH-mediated reduction in macrophage lipid burden and to obtain the final proof that lack of CEH will be atherogenic. Therefore, it is the central hypothesis of this proposal that: Macrophage CEH levels determine intracellular CE accumulation and thereby influence atherosclerotic lesion development as well as systemic aberrations associated with lipid burden of artery wall associated macrophage foam cells. The following three specific Aims are proposed: Aim 1: To determine the role of macrophage-specific CEH expression in reducing the progression of existing atherosclerotic lesions in LDLR-/- mice; Aim 2: To determine the systemic effects of CEH-mediated reduction in Macrophage cholesterol burden in LDLR-/-CEHTg mice and delineate the underlying mechanisms and Aim 3: To obtain in vivo proof of concept by macrophage-specific targeted disruption of CEH in mice and to determine its effect on intracellular CE metabolism and atherosclerosis. Formation of macrophage foam cells is one of the initial events in atherogenesis. HDL- mediated efflux of unesterified or free cholesterol (FC) from the stored cholesteryl esters (CE) in these foam cells is the primary mechanism for removal of intracellular cholesterol and foam cell regression. Extensive earlier research has indicated aberrations in macrophage cholesterol ester metabolism in animal species susceptible to atherosclerosis. Further, these studies have also identified intracellular cholesterol ester hydrolysis catalyzed by neutral cholesteryl ester hydrolase (CEH) as a limiting step in the process of cholesterol efflux. During the last funding cycle of this project we demonstrated the role of macrophage CEH in regulating cellular CE levels, enhancing FC efflux and thereby attenuating diet-induced atherosclerosis in LDLR-/- mice. The proposed studies will build on this foundation and establish the role of CEH in not only preventing progression of existing plaque but in improving systemic inflammation and insulin sensitivity as a result of decreasing macrophage lipid burden. Given the prevalence of atherosclerosis and coronary artery disease with obesity and insulin resistance as major risk factors, the current findings are likely to have important clinical relevance.